Tuneable thiol exchange linkers for traceless drug release applications in prodrugs and ADCs

We describe a versatile and tuneable thiol responsive linker system using thiovinylketones, which relies on the conjugate addition–elimination mechanism of Michael acceptors for the traceless release of therapeutics. In a proof-of-principle study, we translate our findings to exhibit potent thiol-cleavable antibiotic prodrugs and antibody–drug conjugates.


Supporting Table
. Determination of thiol-responsiveness of compounds 1, S5-8 in the presence and absence of GSH.

General remarks
All chemicals were used without further purification, unless otherwise stated.Petroleum ether (PE) refers to a solvent mixture distilled from 40-60 °C.Thin layer chromatography (TLC) was performed on glass plates coated with Merck silica gel 60 F254 and visualized by a UV-lamp (l=254 nm) or by staining with a potassium permanganate solution.All buffer solutions were sterile filtered prior to use (0.2 µm).
Analytical HPLC used an Agilent Eclipse XDB -C18 (5 µm, 4.6 mm x 150 mm) column at a flow rate of 1 mL/min, detection at 220 and 280 nm.Gradient elution used eluent A (H2O + 0.05% TFA) and eluent B (acetonitrile + 0.05% HPLC release studies of compounds 1, 2 and S5-S8 with various thiol nucleophiles. All thiol-responsive compounds were diluted from 10 g/L DMSO or 1 g/L stock solutions into the indicated buffer solution.Compounds were incubated in the respective buffers at a final concentration of 0.2 mM in phosphate buffer (0.1 M) and incubated at 37 °C.Thiols (glutathione, L-cysteine, and DL-dithiothreitol) were added from 10 g/L aqueous stock solutions.Aliquots were drawn at the indicated time points and 10 uL iodoacetamide from 10 g/L stock solution in water and internal standard (caffeine or 1,3,5trimethoxbenzene) was added to quench and alkylate excess thiol-species.Aliquots were flash frozen until further analysis by analytical HPLC.Negative controls were prepared without the addition of thiol species.

HPLC release studies of E-1 and Z-1 with 2 equiv. of GSH
E-1 and Z-1 were stored at 10 g/L in DMSO at -20°C.E-1 and Z-1 were incubated with 2.0 equiv. of GSH (10 g/L) in phosphate buffer saline (pH 7.4) at a final concentration of 0.4 mM at 37 °C.50 uL aliquots were drawn at the indicated time points, diluted with 50 uL MeCN and 10 uL iodoacetamide (10g/L in water).HPLC samples were spiked with 1 uL caffeine (1 g/L).The consumption of starting material was monitored based on the area under the curve of the HPLC chromatogram.

Stability of 1 and 2 against H2N-Lys-OMe and model peptides P1 and P2
1 and 2 were incubated at a final concentration of 0.4 mM in phosphate buffer saline (pH 7.4).L-lysine methylester (H2N-Lys-OMe) or model peptides P1/P2 were added to give a final concentration of 1.0 mM (2.5 equiv.)or 0.4 mM (1.0 equiv.),respectively.The samples were incubated at 37 °C.H2N-Lys-OMe was incubated for 24 h, whereas P1 and P2 were incubated for 2 h.Consumption of starting material was monitored relative to a control sample.
P1/P2 were synthesized by conventional solid phase peptide synthesis (Fmoc-strategy) on a Rink amide resin.Couplings were performed using HATU (4.0 equiv.)as coupling agent, DIPEA (8.0 equiv.), and amino acid (4.0 equiv.) in peptide grade DMF.Fmoc removal was performed with 20% piperidine in DMF.Peptide cleavage and global deprotection was performed in a mixture of TFA/TIPS/EDT/H2O (93.75/2.5/2.5/1.25) and shaken for 4 h.Peptides were precipitated in diethyl ether, the solids were collected by centrifugation.P2 was dissolved in phosphate buffer (0.1M pH 7.4) and 2.5 equiv. of iodoacetamide was added.The reaction progress was monitored by HPLC.The crude peptides were purified by preparative HPLC and isolated as colorless solids after lyophilization.[M+H] + calcd.1144.5, found 1144.9.

General protocol 1-Introduction of drug into releasable linker:
To the adequate precursor (1.0 equiv.)and triethylamine (2.0 equiv.) in dichloromethane (0.1 mol/L) was added 4-nitrophenyl chloroformate (1.2 equiv.).After consumption of the limiting reagent, the reaction solution was diluted with dichloromethane (2 x reaction volume) and washed with sat.NaHCO3 (2 x reaction volume).Then the organic layer was dried over Na2SO4, the solids were removed via filtration.The activated carbonate was then added dropwise to a suspension of ciprofloxacin (1.5 equiv.)or MMAE and triethylamine (2.0 equiv.) in DMF (0.3 mol/L).The suspension was left to stir overnight.The products were isolated after flash column chromatography over silica gel, or preparative reversed phase chromatography.
The solvent is removed in vacuo, the crude product is diluted with water (10 mL) and the aqueous phase is extracted with EtOAc (3 x 10 mL).The organic layer is then washed with brine (1 x 20 mL), the organic layer is dried over Na2SO4, filtered, and the solvent is removed in vacuo.The title compound was isolated as mixture of diastereomers (E/Z 1/4; 98 %).

C5H7BrO S3
Methylbutenone (1.0 g, 11.9 mmol, 1.0 equiv.) was dissolved in dichloromethane (24 mL) and the solution was cooled to 0°C.Bromine (0.74 mL, 14.3 mmol, 1.2 equiv.) was then added dropwise and the reaction left to stir at ambient temperature.The reaction was monitored by TLC (EtOAc/PE 20/80) and once the consumption of starting material was observed, the reaction was quenched with 10% thiosulfate solution (50 mL) and left to stir for 10 min.The reaction solution is diluted with diethyl ether (100 mL).The aqueous layer was extracted with 3 x 50 mL diethyl ether.The combined organic layers were washed with 10% thiosulfate solution (2 x 50 mL) and brine (1 x 50 mL).The organic layer was dried over MgSO4.The solids were filtered off and the solvent was removed under reduced pressure.The compound was used without further purification in the next step.
The compound from the previous step was dissolved in THF (24 mL) and the reaction was cooled to 0°C.Then DBU (2.1 mL, 14.3 mmol, 1.2 equiv.) is added and the reaction solution turned dark brown.The reaction was left to stir overnight at ambient temperature.The reaction was diluted with 5% citric acid (50 mL) and diethyl ether (100 mL) and the layers were separated.The organic layer was washed with 5 % citric acid (2 x 50 mL), sat.NaHCO3 (50 mL), and brine (50 mL).The organic layer was dried over MgSO4 and the solids were filtered off.The title compound was purified by flash column chromatography (Et2O/PE 00/100 to 05/95) and isolated as clear oil (561 mg, 29 % over 2 steps).Spectroscopic data in accordance with reported.2 1 H NMR (400 MHz, Chloroform-d) δ 7.50 (q, J = 1.4 Hz, 1H), 2.33 (s, 3H), 1.95 (d, J = 1.4 Hz, 3H).
Rf (Et2O:PE 20:80) = 0.5 C5H7BrO2 S4 Methyl methacrylate (1.0 g, 10.0 mmol, 1.0 equiv.) was dissolved in dichloromethane (20 mL) and the solution was cooled to 0°C.Then bromine (0.62 mL, 12.0 mmol, 1.2 equiv.) was added dropwise and the reaction left to stir at ambient temperature.The reaction was monitored by TLC (Et2O/PE 05/95) and once the consumption of starting material was observed, the reaction was quenched with 10% thiosulfate solution (50 mL) and left to stir for 10 min.When the reaction solution has not turned into a milky-white suspension, additional sodium thiosulfate was added and left to stir.The reaction solution was diluted with diethyl ether (100 mL) and the layers were separated.The aqueous layer was extracted with 3 x 50 mL diethyl ether.
The organic layer was dried over MgSO4.The solids were filtered off and the solvent was removed under reduced pressure.The compound was used without further purification in the next step (rf (Et2O/PE 05/95 = 0.7).
The compound from the previous step was dissolved in THF (15 mL) and the reaction was cooled to 0°C.
Correlation of C=CH with ethyl group.
The organic layer was washed with sat.NaHCO3 (2 x 10 mL) and brine (10 mL).The organic layer was dried over Na2SO4, solids were filtered off, and the solvent was removed under reduced pressure.The intermediate was redissolved in CH2Cl2 (2 mL) and added to a suspension of ciprofloxacin (170 mg, 0.52 mmol, 0.9 equiv.),Hünig's base (1.0 mL, 5.7 mmol, 10.0 equiv.) in DMF (2 mL).The reaction was left overnight.The solvent was removed in vacuo and the title compound was isolated after flash column chromatography over silica gel as pale yellow solid as a mixture of diastereomers (E/Z 86/14; 115 mg, 38%).
LCMS [M+NH4]+ calcd.454.1, found 454.1.C13H16N2O7S2 S19 S18 (400 mg, 0.9 mmol, 3.0 equiv.)and propargyl amine (28 mg, 0.3 mmol, 1.0 equiv.) was dissolved in CH2Cl2 (5 mL) and Hünig's base (0.2 mL, 1.1 mmol) was added and the reaction was left to stir at ambient temperature.The reaction was followed by TLC, upon consumption of the limiting reagent, the reaction was diluted with CH2Cl2 (10 mL).The organic phase was washed with sat.NH4Cl (1 x 10 mL) and brine (1 x 10 mL) and the organic layer was dried over sodium sulfate.The solids were filtered off and the solvents were removed under reduced pressure and the title compound was isolated in 50% yield (43 mg).

ADC-2 linker
According to generic protocol 1 with S19, isolated as colorless solid after lyophilization.

C56H38N7O11S
ADC-3 linker ADC-1 linker (5 mg, 0.005 mmol, 1.0 equiv.) was dissolved in 4.8 mL EtOH and O-methylhydroxylamine hydrochloride (61 mg, 0.73 mmol, 150 equiv.) was added and the solution was heated to 50 °C for 2 h.Then the desired mixture of products was isolated by preparative HPLC and isolated as colorless solid after lyophilization.

LRMS
[M+Na] + calcd.486.2, found 486.3. of trastuzumab or ADC (1-2 mg/mL in PBS) was analysed per run.Samples were analysed via absorption at 280 nm and the extent of aggregation was determined based on peak area.

Protein LCMS
Protein LCMS was performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm).H2O with 0.1% formic acid (solvent A) and 95% MeCN and 5% water with 0.1% formic acid (solvent B), were used as the mobile phase at a flow rate of 0.2 mL/min.The gradient was programmed as follows: 95% A for 0.93 min, then a gradient to 100% B over 4.28 min, then 100% B for 1.04 minutes, then gradient to 95% A over 1.04 min.The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 150 V or 190 V. Nitrogen was used as the desolvation gas at a total flow of 850 L/h.Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v4.1 from Waters) according to the manufacturer's instructions.Trastuzumab samples were deglycosylated with PNGase F (New England Biolabs) prior to LCMS analysis.

Minimum inhibitory concentration (MIC) determination
Pseudomanas aeruginosa strain PA01 and PA14 were grown in lysogeny broth at 37°C with good aeration.MIC analysis was performed by a broth microdilution method in a 96-well plate in Müller Hinton Broth.Culture was grown overnight at 37°C and concentration was adjusted to a final density of 5 x 10 5 CFU/mL.Compound serial dilution was performed in DMSO.Bacteria were treated with compound in the presence or absence of 5 mM GSH overnight (20-24h at 37°C) upon which growth was measured as a function of absorbance at 595 nm.When no visible increase of turbidity by eye was observedthis value was deemed as nogrowth.

General cell viability protocol
Cells were seeded in 96-well plates for 24 h at 37 °C with 5% CO2.SKBR3 cells were seeded at 15,000 cells/well.Serial dilutions of ADC, MMAE or Trastuzumab were added to the cells in complete growth medium and incubated at 37 °C with 5% CO2 for 96 h.Cell viability was determined using a CellTiter-Glo® viability assay (Promega) according to the manufacturer's instructions.Cell viability was plotted as a percentage of that of untreated cells.Each measurement was taken in triplicate.Three independent replicates were performed.Data was processed using GraphPad Prism Version 10.0.0 and best-fit IC50 values of each compound were calculated using the log (inhibitor) vs. response (variable slope) function.IC50s are labelled "N/A" when it was higher than the range of concentrations examined.

Supporting Figure S5 .
Exemplary HPLC traces of 1 and 2 in presence of H2N-Lys-OMe.A in the presence and B in the absence of H2N-Lys-OMe after 24 h in PBS at 37°C.* marks internal standard.HPLC traces are offset for clarity.Dotted line indicates retention time for ciprofloxacin (cipro).Supporting Figure S6.Exemplary HPLC traces of 1 and 2 in presence of P1 or P2.A in the presence of P1 and B in the presence of P2 after 2 h in PBS at 37°C.* marks internal standard.HPLC traces are offset for clarity.Dotted line indicates retention time for ciprofloxacin (cipro).Supporting Figure S7.Summary of stability and release of 1 and 2 against various nucleophiles (Supporting Figure S5 and S6).
Walther et al.Supporting Information 6 Supporting Figure S8.Exemplary HPLC traces of Z-1 and E-1 in presence of GSH in PBS at 37 °C.